This goal of this component of the U19 cooperative agreement is to create an optimized HIV-1 microbicidal product based on Lactic Acid Bacteria (LAB) that have been genetically engineered to secrete the virucidal compound cyanovirin (CV-N), a naturally occurring protein purified from extracts of cultured cyanobacterium (Nostoc ellipsosporum). CV-N exerts its potent antiviral activity by binding to high mannose residues in the HIV-1 viral envelope and prevents virus entry by blocking fusion with the cell membrane. CV-N retains antiviral activity at nanomolar concentrations against a broad range of HIV-1 isolates in vitro. Application of CV-N in gel form to the rectum and vagina prevents SIV transmission in animal models. Our rationale for choosing strains of LAB as mucosal delivery vehicles for CV-N derives from the well-accepted bio-safety profile of LAB. Recent technical advances allow bioengineering of LAB to secrete heterologous proteins. Some strains of LAB colonize mucosal surfaces after a single administration. Thus, LAB based microbicides may potentially be administered well before sexual contact and serve as an in situ locus of mucosal microbicide production. Finally, certain strains of LAB produce hydrogen peroxide (H2O2), a known antimicrobial compound that exerts anti-viral and anti-bacterial activity in the cervicovaginal tract. We have bio-engineered different strains of LAB to secrete CV-N. HIV-1 challenge assays demonstrate the antiviral potency of LAB secreting CV-N (LAB-CV-N) against laboratory and primary patient derived isolates of HIV-1. In conjunction with other components of this cooperative agreement, we will expand upon our promising preliminary work and pursue the following aims. 1. We will first identify optimum microbicidal strains of bioengineered LAB that adhere to and colonize vaginal/rectal mucosa, produce hydrogen peroxide and are able to secrete CV-N. These microbicidal strains will be tested for their capacity to induce cytotoxicity using a cervical organ culture model. 2. We will further manipulate existing CV-N expression cassettes to optimize CV-N secretion. 3. We will administer LAB secreting CV-N to the vagina and rectum of pigtailed macaques and characterize mucosal colonization and levels of CV-N secretion. In sum, the studies outlined will lead to the development and thorough in vitro and in vivo testing of a novel HIV-1 microbicide.